The invention relates to the field of molecular biology and microbiology. More specifically, a method has been developed whereby In vitro transposition is used to identify chromosomally integrated nucleic acid molecules having unknown function.
With the advent of large-scale genome sequencing efforts, enormous amounts of sequence information is being made available to the research community on a daily basis. Genome sequencing efforts have been completed for the eukaryotes Saccharomyces cerevisiae and Caenorhabditis elegans and for several prokaryotes including Esherichia coli, Mycoplasma gentalium, Bacillus subtilus, Thermotoga maritima, Methanococcus jannaschii, including those of pharmaceutical interest. In spite of the volume of sequence data now available, only a small portion of the genes sequenced today from all these efforts have been functionally characterized.
The need to discover gene function has spawned a new area of research now referred to as xe2x80x9cfunctional genomicsxe2x80x9d. Functional genomics seeks to discover gene function with only nucleotide sequence information in hand. A variety of techniques and methods have been employed in this effort including the use of gene chips, bioinformatics, disease models, protein discovery and expression, and target validation. The ultimate goal of many of these efforts has been the development of high-throughput screens for genes of unknown function.
Techniques applied to elucidate gene function include identifying the interacting protein partner of a gene product as in the yeast 2-hybrid system (Bolger, G., Methods Mol Biol. (Totowa, N.J.) (1998), 88, (Protein Targeting Protocols), 101-131) and transposon tagging, which is useful in both microbial and eukaryotic genomes (Kumar et al., Plant Biotechnol. (Tokyo) (1998), 15(4), 159-165). The logarithmic increase in sequence data has driven the need for high-through-put (HTP) functional genomics screens. However, relatively few HTP methods have been developed to date. Traditional methods for the determination of gene function still remain the basis of the functional genomics effort.
Classically, the first and most basic analysis for any gene is the creation of a null mutation to assess the phenotype of the organism when the gene of interest is rendered nonfunctional. These null mutations are often produced by gene disruption (also called gene knockout or gene replacement) using gene disruption vectors produced by recombinant DNA techniques. Upon transformation into the organism the DNA construct with disrupted gene integrates at the resident location in the genome by homologous recombination and replaces the functional copy of the gene with the nonfunctional gene disruption vector. Gene disruption vectors are constructed from a genomic clone containing the gene of interest.
The above methods have worked well for disruption of genes in a range of organisms but have several inherent limitations, including being limited to knowledge of restriction sites, the unpredictable effects of co-suppression or gene silencing as well as being time consuming and labor intensive. More rapid methods that are adaptable to high throughput screening are needed for the functional analysis of gene function.
Transposons have proven to be invaluable genetic tools for molecular geneticists. Several uses of transposon include mutagenesis for gene identification, reporter libraries for analysis of gene expression, DNA sequencing for relative gene positioning on genetic maps, etc. Until recently, however, all of these applications involved the use of in vivo transposition reactions. However, the commercialization of several In vitro transposition reactions for DNA sequencing and mutagenesis could lead to the replacement of these more traditional in vivo methodologies with more efficient biochemical procedures.
The use of In vitro transposition for the mutagenesis of specific genes was first reported by Gwinn et al. (Journal of Bacteriology, (1997) (Washington, D.C.), vol. 179, no. 23, p. 7315-7320). In their work, the genomic DNA from the naturally transformable microorganism (Haemophilus influenzae) was mutagenized using the Tn7 In vitro transposition system. DNA sequencing using primers that hybridize to the end of the transposon identified mutations in the genes resulting in a reduced expression of constitutive competence genes.
Reich et al. (Journal of Bacteriology, (1999) (Washington, D.C.), vol. 181, no. 16, p. 4961-4968) used the Ty1-based transposition system (Primer Island) to scan the entire Haemophilus influenzae genome for essential genes. The putative essential genes were identified by two methodsxe2x80x94mutation exclusion and zero time analysis. Mutational exclusion involves the identification of open reading frames that do not contain transposon insertions. Zero time analysis involves the monitoring the growth of individual cells after transformations over time; cells containing transposon insertions into-essential genes will be lost over time.
However, to date the use of In vitro transposition for making chromosomal mutations has been limited to the naturally transformable microorganisms (e.g., Haemophilus influenzae). Since most microorganisms are not naturally transformable, methods for making random chromosomal mutations in all microorganisms in a high-throughput manner is needed. Because the above two systems use linear DNA in the transformations, single-crossover events cannot be obtained. Thus, the above systems are not useful to making mutations in essential genes that are involved in cell survival. Another limitation is that the above systems cannot be used to determine the functions of unknown genes on a genomic scale.
The present invention solved the problems by providing a method to make random mutations in genomic scale and screen for essential genes that are responsible for the specific phenotype.
The present invention provides a method for the identification of an essential gene responsible for the presence of a specific phenotype in a recombination proficient microorganism comprising:
a) contacting In vitro:
(i) a transposable element comprising at least one first genetic marker;
(ii) a transposase for the insertion of the transposable element into the essential gene; and
(iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
xe2x80x83under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
b) cloning the transposon-disrupted gene into a suitable vector to form a chromosomal integration vector, said vector comprising at least one second genetic marker;
c) transforming a recombination proficient microorganism which is not naturally transformable with the chromosomal integration vector of step (b) to create transformants;
d) selecting the transformants of step (c) under conditions whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant, by identifying transformants expressing the first genetic marker;
e) culturing the identified transformants of step (d) under conditions whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
f) selecting transformants of step (e) which express either the first genetic marker or both the first and second genetic markers by which transformants having undergone either a single or double crossover event are identified;
g) screening the transformants of step (f) which have undergone either a single or double crossover event, for the presence of a specific phenotype wherein the transformants which are positive for the specific phenotype contain a transposon disrupted gene; and
h) isolating the transposon disrupted gene from the transformant of step (g) which is positive for the specific phenotype.
In one embodiment step (d) of the above described method is optionally deleted.
Additionally the method may comprise using a temperature sensitive chromosomal integration vector which will integrate into the host genome at a non-permissive temperature. Thus the invention provides a method for the identification of an essential gene responsible for the cell growth under any condition in a recombination proficient microorganism comprising:
a) contacting In vitro:
(i) a transposable element comprising at least one marker;
(ii) a transposase for the insertion of the transposable element into the essential gene; and
(iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
xe2x80x83under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
b) cloning the transposon disrupted gene into a temperature sensitive vector containing a second genetic marker to form a temperature sensitive chromosomal integration vector;
c) transforming a recombination proficient microorganism, which is not naturally transformable, with the temperature sensitive chromosomal integration vector of step (b) to create transformants;
d) culturing the transformants of step (c) at a permissive temperature whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
e) identifying transformants of step (d) expressing the marker;
f) culturing the identified transformants of step (e) at non-permissive temperatures whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
g) selecting transformants of step (e) which did not grow at step (f) after chromosomal integration between the chromosomal integration vector and the genome of the transformant and which contain a transposon disrupted gene; and
h) isolating the transposon disrupted gene from the transformant of step (g) which is responsible for cell growth under any condition.
In one embodiment a third genetic marker may be used. Thus the invention additionally provides a method for the identification of an essential gene responsible for the presence of a specific phenotype in a recombination proficient microorganism comprising:
a) contacting In vitro:
(i) a transposable element comprising at least one first marker;
(ii) a transposase for the insertion of the transposable element into the essential gene; and
(iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism;
xe2x80x83under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene;
b) cloning the transposon disrupted gene into a suitable vector to form a chromosomal integration vector, said vector comprising at least one second marker and at least one third marker;
c) transforming a recombination proficient microorganism which, is not naturally transformable, with the chromosomal integration vector of step (b) to create transformants;
d) selecting the transformants of step (c) under conditions whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant, by identifying transformants expressing the first marker;
e) culturing the identified transformants of step (d) under conditions whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant;
f) selecting transformants of step (e) which express either the first marker alone, the first and second markers alone, or the first, second and third markers, by which transformants having undergone either a single or double crossover event are identified;
g) screening the transformants of step (f) which have undergone either a single or double crossover event, for the presence of a specific phenotype wherein the transformants which are positive for the specific phenotype contain a transposon disrupted gene; and
h) isolating the transposon disrupted gene from the transformant of step (g) having the specific phenotype.
Genetic markers used in the present method may be selectable or screenable and may incorporate genes useful for the construction of positive selection vectors, such as the sacB gene of Bacillus. In a preferred embodiment the first and second genetic markers are different.